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  • Title: Identification of two functional estrogen response elements complexed with AP-1-like sites in the human insulin receptor gene promoter.
    Author: García-Arencibia M, Dávila N, Campión J, Carmen Carranza M, Calle C.
    Journal: J Steroid Biochem Mol Biol; 2005 Feb; 94(1-3):1-14. PubMed ID: 15862945.
    Abstract:
    This study was designed to explore the possible existence and location of estrogen response elements (EREs) in the human insulin receptor (hIR) gene promoter. Transfections of U-937 cells with the reported plasmids phIR(-1819)-GL2, phIR(-1473)-GL2, and phIR(-876)-GL2, that contain the -1819 to -271 bp fragment of the hIR promoter (wild-type promoter) and progressive 5' deletions of this promoter, revealed that while the activity of the wild-type promoter, was repressed 36% by treatment with 17beta-estradiol (E(2)), the activities of progressive 5' deletions of this promoter were reduced by 26% and by 0%, by this hormone. This suggests that E(2) needs the wild-type promoter for full transcriptional repression of this gene and it also suggests the presence of putative EREs in the region between -1819/-877 bp of this promoter. To identify these EREs we performed a computer search, using the SEQFIND programme developed in our laboratory, by homology with the consensus vit-ERE (5'GGTCAnnnTGACC3') of the Xenopus vitellogenin A(2) gene promoter. The results of our search indicated no sequence identical to this consensus ERE, and neither was any sequence found to show 9 or 8 of the 10 bases of this consensus in this promoter. Nevertheless, a putative hIR ERE1 (5'AGTGAaacTGGCC3') showing 7 bases of the consensus vit-ERE, and 10 bases of the optimal binding sequence ERE (5'CA/GGGTCAnnnTGACCT/CG3'), was identified between -1430/-1418bp of the hIR promoter. An AP-1-like site was covering the 3' half-element of this ERE; another AP-1-like site was overlapping the first AP-1-like site, and finally a third AP-1-like site was located beside to the 5' half-element. In addition, another putative hIR ERE2 (5'GCTCCtagCAAAC3') showing 5 bases of the consensus vit-ERE, and 9 bases of the optimal binding sequence ERE, was located upstream of the hIR promoter, between -1567/-1555 bp. An AP-1-like site was located downstream of the 3' half-element of this ERE, and another AP-1-like site was beside the 5' half-element. EMSA analysis using nuclear extracts of E(2)-treated cells and natural sequences, including these putative EREs, indicated that ERbeta - the only isoform expressed in U-937 cells - specifically recognized both EREs because ERbeta-DNA complexes were efficiently competed by the corresponding unlabelled probe and supershifted by the anti-human ERbeta (L-20) antibody. These data provide the first identification of EREs complexed with AP-1-like sites in the hIR promoter, which account for the transcriptional repression of the hIR gene mediated by ERbeta in U-937 cells.
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